Topical microbicides that block transmission of herpes simplex virus (HSV) and other sexually transmitted pathogens are urgently needed. The observation that heparan sulfate proteoglycans serve as attachment receptors propelled the development of sulfonated polymers, which are now in clinical trial, as candidate microbicides. Identification of new agents that target steps post-binding requires elucidation, at a molecular level, of the signaling pathways required for fusion of the viral envelope with the cell plasma membrane and nuclear transport of viral capsids. Preliminary studies indicate that viral invasion requires activation of calcium (Ca2+) signaling and tyrosine phosphorylation of focal adhesion kinase (FAK) and other cellular proteins. Activation of Ca2+ and phosphorylation signaling pathways are common mechanisms associated with invasion by other microbes, most notably HIV. The studies proposed in this application build on these observations. Focusing on HSV serotype 2 (HSV-2), the predominant cause of genital herpes, these studies will examine the cellular signaling pathways required for viral invasion and the role viral envelope glycoproteins play in this process. Most studies of viral entry have been conducted using permanent non-polarized epithelial cell lines. However, the polarity of epithelial cells is fundamental to their structure and function and influences susceptibility to infection. Preliminary work indicates that HSV- 2 infection of polarized epithelial cells is more efficient following apical compared to basolateral exposure. Therefore, the mechanism of viral invasion in polarized cell culture systems will also be explored. Observations in polarized cells will more accurately reflect the conditions in vivo. Results of these studies will enhance understanding of the molecular biology of HSV invasion and should facilitate the rational design of new topical antimicrobials to prevent HSV transmission and acquisition.